Zuidam N J, Hirsch-Lerner D, Margulies S, Barenholz Y
Department of Biochemistry, The Hebrew University-Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel.
Biochim Biophys Acta. 1999 Jul 15;1419(2):207-20. doi: 10.1016/s0005-2736(99)00069-3.
Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was </=0.5, at which only 10-30% of DOTAP in the lipoplex is neutralized. Prolonged incubation time of lipoplexes before addition to cells slightly decreased the level of transfection. A major influence on the lipofection level was found when the mode of lipoplex preparation was varied. Mixing plasmid DNA and DOTAP/DOPE (1:1) LUV in two steps instead of one step resulted in a higher lipofection when at the first step the DNA/DOTAP mole ratio was 0.5 than when it was 2.0. Only static light-scattering measurement, which is related to particle size and particle size instability, revealed differences between the lipoplexes as a function of lamellarity of the vesicles (MLV or LUV), mixing order, and number of mixing steps. Other physical properties of these lipoplexes were dependent only on the DNA/DOTAP mole ratio, i.e. the extent of DOTAP neutralization (as monitored by ionization of the fluorophore 4-heptadecyl-7-hydroxycoumarin) and the extent of defects in lipid organization (as monitored by level of exposure of the fluorophore 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene to water). The secondary and tertiary structure of DNA in lipoplexes was evaluated by circular dichroism spectroscopy. The results of this study point out that the structure of lipoplexes should be physicochemically characterized at two different levels: the macro level, which relates to size and size instability, and the micro level, which relates to the properties described above which are involved in the intimate interaction between the plasmid DNA and the lipids. At the micro level, all parameters are reversible, history-independent and are determined by DNA/DOTAP mole ratio. On the other hand, the macro level (which is the most important for transfection efficiency) is history-dependent and not reversible.
研究了由N-(1-(2,3-二油酰氧基)丙基)-N,N,N-三甲基氯化铵（DOTAP）组成的脂质体与或不与辅助脂质复合的人生长激素表达载体对NIH-3T3细胞的转染情况。转染效率取决于用于制备脂质体复合物的脂质体的层状结构。无论脂质组成如何，多层囊泡（MLV）比约100nm的大单层囊泡（LUV）更有效。转染的最佳DNA/DOTAP摩尔比≤0.5，此时脂质体复合物中只有10-30%的DOTAP被中和。脂质体复合物在加入细胞前延长孵育时间会略微降低转染水平。当改变脂质体复合物的制备方式时，发现对脂质体转染水平有重大影响。分两步而不是一步混合质粒DNA和DOTAP/DOPE（1:1）LUV，当第一步DNA/DOTAP摩尔比为0.5时比为2.0时产生更高的脂质体转染。只有与粒径和粒径不稳定性相关的静态光散射测量揭示了脂质体复合物之间因囊泡（MLV或LUV）的层状结构、混合顺序和混合步骤数而产生的差异。这些脂质体复合物的其他物理性质仅取决于DNA/DOTAP摩尔比，即DOTAP的中和程度（通过荧光团4-十七烷基-7-羟基香豆素的电离监测）和脂质组织缺陷程度（通过荧光团1-(4-三甲基铵苯基)-6-苯基-1,3,5-己三烯暴露于水的水平监测）。通过圆二色光谱法评估脂质体复合物中DNA的二级和三级结构。本研究结果指出，脂质体复合物的结构应在两个不同水平上进行物理化学表征：宏观水平，与粒径和粒径不稳定性有关；微观水平，与上述参与质粒DNA与脂质之间紧密相互作用的性质有关。在微观水平上，所有参数都是可逆的、与历史无关的，并且由DNA/DOTAP摩尔比决定。另一方面，宏观水平（对转染效率最重要）是与历史有关的且不可逆的。
